| Summary: | 碩士 === 國立陽明大學 === 臨床牙醫學研究所 === 94 === Areca nut quid chewing is a common habit of Asians. There are many cytotoxic or genotoxic integrants releasing from the quid during the chewing course. Although there are still no enough evidences can prove areca nut quid chewing leads to cause periodontal diseases yet at present. According to the datas of epidemiological studies, there is higher prevalence of periodontal diseases among areca nut quid chewers than non-areca nut quid chewers. In addition, several in vitro studies suggest that components of areca nut quid are toxic to gingival keratinocytes, gingival fibroblasts and periodontal ligament fibroblasts. At the same time, components of areca nut quid may reduce bactericidal activity of neutrophils. But little is known about the effects of components of areca nut quid on osteoblasts, one of the cellular components of periodontal tissue. The purpose of this study is to discover the effects of ripe areca nut extract (rANE) and arecoline on the human osteoblast-like cell line (MG63). First, morphology of MG63 cell was observed with inverted microscope. Then cytotoxic effects of rANE and arecoline on MG63 cell were analysised with MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) colorimetric assay. Moreover, the expression of genes which associated with bone metabolism, included procollagen α1 of type I collagen (COLIα1), alkaline phosphatase (ALP), osteocalcin (OC), osteoprotegerin (OPG), receptor activator of nuclear factor κB ligand (RANKL) were examined with reverse transcriptase polymerase chain reaction (RT-PCR). Furthermore we utilized ALP colorimetric assay and fluorescence immunoassay (FIA) to explore the ALP activity and the amounts of RNAKL protein, respectively. In addition, Genes of MG63 cell with the difference on displaying before and after stimulated by rANE were screening with cDNA microarray analysis. Stimulated MG63 cell with rANE or arecoline separately, found that both were cytotoxic to MG63 cell, among them rANE was relatively more cytotoxic to MG63 cell. In addition, the results showed that rANE caused reduce the mRNA expressions of COLIα1 and ALP, promoted the mRNA expressions of RANKL and OC. Arecoline induced suppression of the mRNA expressions of COLIα1 and RANKL, caused increase of the mRNA expressions of ALP and OC. We found that ALP activity and the amounts of RNAKL protein of MG63 cell were dropped or stimulated after stimulated by rANE, respectively. The results of cDNA microarray analysis pointed out the expression of four gene which related to bone resorption were increased, include: matrix metalloproteinase 1 (MMP1), interleukin 1 receptor, the behavior of protein kinase C (PKC ), tumor necrosis factor receptor superfamily. This thesis found that rANE and arecoline both were cytotoxic to MG63 cell by in vitro study. Among them, the effects of rANE on MG63 cell, included reduced ALP activity, rised amounts of RANKL protein and genes related to bone resorption, were all incline to stimulate the bone metabolism to resorption.
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